The invention concerns growth-inhibited hydroxyapatite (referred to in the following as wHAP) for the improvement of bone healing. It differs from the apatites used already up to now in that it releases calcium and phosphate ions in physiological solutions and, in contrast to customary hydroxyapatites, does not bind them. Thereby it promotes new bone formation and bone growth.
The development of new biomaterials is primarily driven by the desire to produce technically mature tissue implants and bone implants for medical applications, However, they are subject to special demands, depending on implantation site, with regard to biocompatibility, mechanical strength, degradation behavior in case of resorbable implants, and bioactivity. The term bioactivity characterizes the ability of a material to support the formation of an apatite layer (bone mineral) on the surface in the presence of natural or simulated body fluid.
The mammalian bone fulfils in the organism supporting functions as well as metabolic functions [Wintermantel, E.; Ha, S. W.: Medizintechnik—Life Science Engineering, Springer Verlag, 2008]. The bone masters the resulting varied demands by its hierarchical construction and by its material composition. The organic bone matrix which makes up approx. 30% of the bone is comprised of up to approx. 95% of collagen. In the course of a mineralization process, mineral calcium phosphate (primarily hydroxyapatite) is deposited between the collagen fibrils in the form of crystalline platelets having a thickness of about two to four nanometers [chapter Forschung aktuell. In: Max-Plank-Forschung (Ed.): Knochen auf den Zahn gefühlt; vol. 1., 2005, pages 10-1]. Depending on the degree of mineralization (in bone approx 65%) the flexible basic scaffold of collagen is strengthened mechanically causing the strength to increase enormously, primarily under pressure load. Based on such findings, for some time now possibilities for the imitation of naturally occurring composite materials, primarily of bone, have been worked on intensively.
In direct bone contact, primarily the degradation behavior decides the destiny of the interacting intravascular or tissue cells in case of resorbable biomaterials. The term degradation behavior is understood in this context as release of substances from the material into the surrounding tissue or the absorption of substances from the surroundings and deposition on the implant surface. In case of the calcium and phosphate balance, the absorption of ions from the surroundings with deposition on the implant surface in the form of an apatite layer is referred to as bioactive behavior. In the field of bone substitution materials it has been reported repeatedly that a high bioactivity can promote the formation of a force-fit connection between implant and recipient tissue. On the other hand, some publications also report negative effects on the cell behavior in connection with a high bioactivity of the biomaterial. From this it can be derived that the ion balance that is determined by the degradation behavior of the biomaterial has a determinative influence on the cell behavior, on the implant surface as well as in the tissue areas close to the implant, and thus on bone remodeling and finally on the success of the treatment.
Materials on the basis of collagen and collagen derivatives are of especially great interest for biomedical applications. As the body's own structural protein it is ubiquitous in all multicellular animals and is the most frequent protein, constituting about one third of the whole protein mass. It is nearly non-toxic, bioresorbable and hardly immunogenic, resulting in an excellent biocompatibility. As a starting material for industrial uses, collagen of the type I is mostly used which can be obtained, for example, from tendons, cartilage and skins of cattle, calves and pigs. Meanwhile numerous products on the basis of collagen have been developed and have been established in many areas of cosmetics and medicine. Gelatin, also established in numerous uses, is a denatured collagen. Since collagen-based products alone as a rule do not match the mechanical requirements as a bone substitute, they are substituted or supplemented with inorganic-non-metallic phases.
In the field of calcium phosphate-based bone substitution materials, these are primarily hydroxyapatite (HAP) and tricalcium phosphate (TCP) as well as mixtures of both phases used [Tadic D, Epple M.; A thorough physicochemical characterization of 14 calcium phosphate-based bone substitution materials in comparison to natural bone. Biomaterials 2004; 25 (6): 987-94]. Hydroxyapatite of biological origin (e.g., the commercially available Endobon, Cerabone) or synthetic origin (e.g., Cerapatite, Ostim) applied in powder form, granular form, block-shaped or pasty form is little bioresorbable [Murugan R, Ramakrishna S., Development of nanocomposites for bone grafting. Comp Sci Tech 2005; 65 2385-406. Hydroxyapatite is considered insoluble in the neutral pH range on account of its composition, crystalline structure and mostly low microporosity. The comparably slow degradation processes in vivo are attributed almost exclusively to cellular resorption [Detsch R A, Mayr H B, Seitz D B, Ziegler G.; Is hydroxyapatite ceramic included in the bone remodeling process? An in vitro study of resorption and formation processes. Key Engineering Materials 2008; 361-363 II 1123-1126], Higher degradation rates can be reached with HAP/TCP mixtures (e.g., 4-Bone, Bonesave) or pure TCP (e.g., Biobase, Cerasorb). However, the degradation behavior of TCP is calculable only with difficulty.
The calcium phosphate phase serves in the natural bone not only for increasing the strength, but is also a calcium supplier in the course of bone metabolism or remodeling. Knowledge of how this occurs in the human organism in the healthy bone is sketchy; in which manner this process intervenes in the healing of defects when degradeable biomaterials are used, is not understood in detail yet and is the object of research. Of special interest is the healing of defects of the osteoporotic bone which occurs, as is known, more slowly. In some publications it has been demonstrated that the calcium concentration in implant surroundings that is affected by the solubility of the implant material has effects on the proliferation and differentiation behavior of stem cells, osteoblasts and osteoclasts [Muller P, Bulnheim U. Diener A, Luthen F, Teller M, Klinkenberg E D, Neumann H G, Nebe B, Liebold A, Steinhoff G, Rychly J.; Calcium phosphate surfaces promote osteogenic differentiation of mesenchymal stem cells. J Cell Mol Med 2008; 12 (1): 281-91]. As described by Detsch et al., a high calcium ion concentration, as obtained e.g. for TCP-based materials, reduces the resorption activity of osteoclasts [Detsch R, Mayr H, Ziegler G.; Formation of osteoclast-like cells on HA and TCP ceramics. Acta Biomater 2008; 4 (1): 139-48]. The most recent publication attributes cytotoxicity in vitro to unsintered hydroxyapatite in the form of a composite of chitosan and HAP, which is explained by reduced calcium or magnesium contents [Malafaya P B, Reis R L. Acta Biomater 2009; 5 644-60].
DE 10 2004 058 893 A1 discloses a method for modifying biopolymers. An example of this process is the use of fibril-forming collagen modified with chondroitin sulfate (CS), wherein upon later implantation the modified collagen reacts with a monomer, glucuronic acid, produced by the metabolic decomposition of the heterodimeric CS present at the collagen. In the course of this reaction, amino acids of the collagen, primarily the ω amino group of lysine, was carboxymethylated causing regions of the collagen surface to become negatively loaded. As a result of the carboxymethylation, calcium ions present in the vicinity are bound. In this way, a heterogeneous nucleus formation of calcium phosphate phases is initiated and the mineral formation, e.g., the biomimetic preparation of HAP in the surroundings of carboxymethylated collagen, is accelerated and intensified.
The carboxymethylated templates are mineralized according to the DCCM or DMDM method. The carboxymethylated templates are fixedly adsorbed on a surface of the employed membranes. In the presence of calcium and phosphate ions the carboxymethylated templates mineralize.
When an supersaturated electrolyte solution with Ca and PO4 ions is present, nuclei of hydroxyapatite form on collagen fibril surfaces at individual selected sites and do not cover the surface evenly and grow together undefined in the course of growth. In this context, FIG. 1a and FIG. 1b show scanning electron microscope images of hydroxyapatite deposited on collagen I fibrils.
FIG. 1a shows the deposition process after 15 minutes in a calcium ion and phosphate ion containing saturated electrolyte solution. In the Figure, the collagen fibrils are easily recognizable in a branched network. On the surfaces of the fibrils mineralization has taken place at selected sites. The hydroxyapatite (HAP) crystallites are easily recognizable by brighter areas in needle-shaped form. The HAP crystallite formation does not occur evenly (heterotactic) on the collagen fibril surface but at some selected preferred sites.
FIG. 1B shows the HAP covering of the surface after an exposure time of two hours. The apatite crystallites have increased in numbers, they penetrate each other mutually and cover the collagen fibrils located underneath irregularly on account of their crystallographic structure.
The growth of the HAP crystallites occurs therefore not epitactically with respect to the collagen surface but heterotactically at individual selected sites of the surface with the crystal appearance that is typical for hydroxyapatite (needle formation). In contrast to living organisms, osteocalcin for nucleus formation is absent so that the mineral formation does not take place in platelet form in the gap regions of the collagen I fibrils. The needle formation is the result of the growth of the crystallites in crystallographically preferred directions. The growth of the needles is interrupted in the preferred direction only when they grow together. The formed crystal surfaces are still capable of growth afterwards. On account of the present surface morphology and surface energy, there is thus growth of other crystal surfaces or formation of new crystallites on the HAP-surface. This fact leads to the formation of a crystal appearance of the hydroxyapatite as it is likewise know from other biological surfaces. These HAP crystals have thus the undesirable property of removing from the electrolyte surroundings calcium as well as phosphate ions causing them to grow and the calcium and phosphate ions to be bound in physiological solutions.
A high bioactivity as it is usually exhibited by unmodified calcium phosphate phases leads, on the one hand, to the formation of an apatite layer which has been positively valued up to now, but then also to the depletion of the surroundings of the biomaterial with respect to calcium ions; this can affect negatively the cells involved in remodeling and thus bone healing or new bone formation. Therefore, one cannot speak of bioactivity in a narrowly defined sense. How high the calcium ion concentration must be in the cell surroundings of osteoblasts or also osteoclasts to create ideal conditions for new bone formation is still unclear up to now and will vary in a wide range. However, it is unambiguously clear that calcium phosphate-based materials are needed whose calcium binding and release behavior can be adjusted in a predetermined way. In this connection, according to the current level of knowledge, metastable calcium phosphate phases, as for example tricalcium phosphate, bruschite, and octacalcium phosphate, are unsuitable because their dissolution behavior is too fast with respect to the bone healing process.
It is therefore the object of the invention to form hydroxyapatites whose growth is inhibited after the nucleus formation and themselves in predetermined way release calcium and phosphate ions after implantation in the bone tissue. The proliferation and the differentiation of bone-forming cells should be influenced positively by this effect and the bone formation should be promoted.